The long range objective of this research is to determine the cellular role of mitochondrial DNA. This will be approached in this particular proposal by studying the mitochondrial RNA polymerase and the RNA gene products obtained by in vitro transcription of the mitochondrial DNA. Closely related studies have been proposed concerning the control of transcription of this genome. Rat liver mitochondrial RNA polymerase has already been partially purified (Reid and Parsons, RNAS, 68, 2830, 1971) and will be further purified to homogeneity. Its properties will be characterized with respect to: 1. its template specificity; 2. its ability to transcribe natural DNA, DNA homopolymers and RNA templates and the nature of this transcription; 3. its ability to carry out nuclease activities; 4. its similarity or lack of similarity to nuclear RNA polymerases, and 5. its molecular weight and possible subunit structure. In addition, the effect of E. coli sigma and rho factors on the transcription will be studied, as well as the nature of the transcription products. Finally, the number of the promoter sites on mitochondrial DNA will be determined. The fidelity of transcription will be determined by base composition and hybridization studies. The ability of the enzyme to nick DNA templates will be studied by determining the P32 incorporation from alpha-P32-ATP into the DNA after incubation with the enzyme and phosphomonoesterase treatment. RNA transcription products will be characterized as to their size by gel electrophoresis and sucrose gradients. The number of DNA promoter sites will be determined by monitoring the incorporation of gamma-P32-nucleotide into DNA in the presence of rifamycin after allowing formation of a DNA-enzyme complex in the absence of rifamycin.